RNA-dependent RNA polymerase 1 from Nicotiana tabacum suppresses RNA silencing and enhances viral infection in Nicotiana benthamiana

Abstract

Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

Figure 1.

Analysis of PPV Infection in the Transgenic N. benthamiana Plants Expressing Nt-RDR1. (A) Protein immunoblot detection of myc-NtRDR1 with anti-c-myc antibody in pools of six T1 plants of different 35S-NtRDR1 and Pro-NtRDR1 transgenic lines. Wild-type N. benthamiana (Nb) was used as negative control. (B) Wild-type control (Nb) or transgenic plants were inoculated with GFP-tagged PPV. Photographs were taken from line 1 and line 7 of 35S-NtRDR1 [each containing one copy of the 35S-Myc-NtRDR1 DNA insert, see Supplemental Figure 1 online, even though line 7, labeled 35S-NtRDR1(-), does not express the transgenic protein] at 25 DAI (top panels) and line 1 of Pro-NtRDR1 at 18 DAI (bottom panels) under UV light to show the GFP expression associated with PPV infection in systemically infected leaves. Local infected leaves were photographed at 4 DAI (insets in top panels). (C) PPV RNA accumulation was examined at similar layers of systemically infected leaves of Nb, Pro-NtRDR1, and 35S-NtRDR1 collected at 10 and 28 DAI. Pools of six plants were analyzed for each plant line. Hybridization was done with a ³²P-labeled PPV antisense cDNA probe. Methylene blue–stained rRNA is shown as loading control. Quantification of PPV-GFP RNA relative to total RNA is shown at the right part of the panel. The value of Nb was arbitrarily designated as 1. (D) Protein immunoblot analysis with anti-c-myc antibody of myc-NtRDR1 induction in Pro-NtRDR1 plants after SA treatment or PPV infection. Pools of four plants were analyzed for each time point. The result of representative line 3 was shown. Coomassie blue-stained total proteins are shown as loading controls.

Figure 2.

Analysis of PPV Infection in RDR6-Silenced (RDR6i) and SA-Deficient (NahG) Transgenic N. benthamiana Plants. (A) GFP-tagged PPV was inoculated to wild-type (Nb), RDR6i, and NahG plants, and photographs were taken at 18 DAI under UV light to show the GFP expression associated with PPV infection in systemically infected leaves. (B) PPV-GFP RNA accumulation was examined at similar layers of systemic infected leaves of Nb, RDR6i, and NahG plants collected at 18 and 25 DAI. The duplicated lanes are two pools of six plants. Hybridization was done as described in Figure 1C. (C) Kinetics of induction of AOX-1 in response to PPV infection. Systemically infected leaves of Nb, NahG, and Pro-NtRDR1 transgenic plants infected with PPV-GFP were collected at different days after inoculation. Pools of four plants were analyzed for each time point. The blot was probed for accumulation of PPV RNA and AOX-1 transcripts using ³²P-labeled specific cDNAs. Methylene blue–stained rRNA is shown as loading control. (D) Detection of RDR1m, RDR2, and RDR6 mRNA accumulation in Nb, 35S-NtRDR1, and RDR6i plants. The RNA gel blots were probed for accumulation of transgene Nt-RDR1 and endogenous RDR6 transcripts using ³²P-labeled specific transcribed RNA. Pools of four plants were analyzed for each plant line. Methylene blue–stained rRNA is shown as loading control. Quantitative real-time RT-PCR for the assessment of RDR1m, RDR2, and RDR6 transcript levels in Nb and Pro-NtRDR1 plants were made on pools of four plants each. Error bars represent standard deviations for three replicates. Relative transcript levels were calculated by the ΔΔC(t) method (Livak and Schmittgen, 2001) using GAPDH transcripts (Schwach et al., 2005) as the internal standard.

Figure 3.

Analysis of the Effect of Nt-RDR1 on Transient Induction of Silencing in GFP-Transgenic 16c N. benthamianaPlants. (A) Leaves of 16c GFP-transgenic plants were coinfiltrated with 35S-GFP and either empty vector, 35S-p19, 35S-myc-NtRDR1, 35S-NtΔRDR1, or 35S-NtRDR1m. Photographs were taken under UV light at 4 dpa. (B) RNA gel blot analysis of GFP mRNA and GFP-derived siRNA accumulation. ³²P-labeled GFP DNA or RNA probes were used. Methylene blue–stained rRNA and U6 RNA hybridization are shown as loading controls. (C) Leaves of 16c, 16c/NtRDR1, and 16c/RDR6i plants infiltrated with 35S-GFP. Photographs were taken under UV light at 4 dpa. (D) RNA gel blot analysis of GFP mRNA and GFP-derived siRNAs accumulation as described in (B).

Figure 4.

Analysis of the Effect of Nt-RDR1 on S-PTGS and Secondary siRNA Synthesis. (A) and (B) Induction of silencing of the GFP transgene in 16c, 16c/NtRDR1, and 16c/RDR6i plants. (A) shows an RNA gel blot analysis of G-, F-, and P-derived siRNA accumulation in 16c, 16c/NtRDR1, and 16c/RDR6i plants infiltrated with 35S-Fi or 35S-F at 4 dpa. ³²P-labeled RNA probes specific for each corresponding GFP portion were used. U6 RNA hybridization is shown as loading control. (B) shows the systemic spread to the whole plant of GFP silencing induced by transient expression of 35S-Fi in 16c and 16c/NtRDR1 plants and the limited silencing spread to one or two upper leaves in a 16c/RDR6i plant. Photographs were taken under UV light at 2 weeks after agroinfiltration. (C) and (D) Induction of silencing of transiently expressed GFP in Nb, Nt-RDR1, and RDR6i plants. (C) shows an RNA gel blot analysis of G-, F-, and P-derived siRNA accumulation in plants coinfiltrated with 35S-GFP and 35S-Fi or 35S-F as described in (A). (D) shows leaves of Nb, Nt-RDR1, and RDR6i plants coinfiltrated with 35S-GFP and 35S-F. Photographs were taken under UV light at 4 dpa.

Figure 5.

Analysis of the Effect of Nt-RDR1 on Small RNA Accumulation and syn-tasiRNA–Mediated Silencing. (A) and (B) Induction of silencing of transiently expressed GFP in Nb, Nt-RDR1, and RDR6i plants coinfiltrated with 35S-173-P/35S-MIR173/35S-GFP. Photographs were taken under UV light at 3 dpa (A). (B) shows RNA gel blot analysis of GFP transcripts, syn-tasiRNAs, and miR173 in Nb, Nt-RDR1, and RDR6i plants at 3 and 6 dpa. ³²P-labeled GFP DNA or oligodeoxynucleotide probes specific for syn-tasiRNA and miR173 were used, respectively. Methylene blue–stained rRNA and U6 RNA hybridization are shown as loading controls. (C) RNA gel blot analysis of endogenous small RNAs in Nb and Nt-RDR1 plants. Oligodeoxynucleotide probes specific for each small RNA were used. U6 RNA hybridization is shown as a loading control. (D) Analysis of vsiRNAs in PPV-GFP–infected Nb and Nt-RDR1 plants. The frequency of each nucleotide at the 5′ end of vsiRNAs of different lengths is indicated.

Figure 6.

Analysis of the Infection of Several Viruses in Wild-Type, Nt-RDR1, and RDR6i Plants. (A) Symptoms of SD-CMV infection in wild-type (Nb), Nt-RDR1, and RDR6i plants. Photographs were taken at 30 DAI. (B) RNA gel blot analysis of viral RNA accumulation in Nb, Nt-RDR1, and RDR6i plants infected with PPV-GFP, CMV, PVX, PVY, TRV-PDS, or TMV-GFP. Total RNA was extracted from inoculated and systemically infected leaves at 4 and 10 DAI. Blots were probed with ³²P-labeled cDNAs specific for the corresponding virus. Methylene blue–stained rRNAs are shown as loading controls. Quantification of viral RNA relative to total RNA is shown at the right part of the panel. The value of Nb was arbitrarily designed as 1.